Cell patterning technique

ABSTRACT

The present invention provides a masking system for selectively applying cells to predetermined regions of a surface. A mask is positioned adjacent to a surface to cover some portions of the surface while allowing other portions of the surface to remain uncovered. Cells then are applied to uncovered portions of the surface and the mask removed. Alternatively, a cell-adhesion promoter is applied to uncovered portions of the surface, and then cells are applied to the surface before or after removal of the mask from the surface. The masking system can be pre-coated, at least on those surfaces which will come into contact with cells, with a cell-adhesion inhibitor to resist absorption of cells and thereby avoid cell damage when the mask is removed (if cells are deposited prior to removal of the mask). A polymeric elastomeric mask that comes into cohesive-conformal contact with a surface to be patterned can be used.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/808,745, filedMar. 15, 2001, entitled METHOD FOR CELL PATTERNING, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/190,399,filed Mar. 17, 2000, entitled CELL PATTERNING VIA AN ELASTOMERIC MASK.

FIELD OF INVENTION

The present invention relates to methods for patterning cells onsubstrate surfaces via an elastomeric mask. These methods allow for thestudy of cell migration and growth.

BACKGROUND OF THE INVENTION

Cell adherence on substrate surfaces, particularly surfaces used forcell-culture such as glass or plastic, is necessary in many instancesfor the study of cells in furthering applications such as tissueengineering, biosensors, etc. Cell patterning, i.e. placing cells indiscrete portions of a surface, has been provided by photolithography.Although the technology of photolithography is very highly developed, itpresents several disadvantages. Photolithography presents harshconditions which can destroy the cells themselves. Clean-room facilitiesand other complex equipment are also required and such facilities andequipment are not readily accessible to most biologists.Photolithography is not amenable to controlling the molecular propertiesof a surface required for many sophisticated cell-biologicalexperiments. In addition, photolithography modifies a surface only atthe beginning of an experiment. Once cells are deposited,photolithography cannot be used to make further surface modifications.

Laminar flow (FLO) patterning involves surface modification via laminarflow of adjacent fluid streams with low Reynolds numbers. FLO patterningis restricted to simple patterning and thus is useful for patterning theenvironment of a cell and for cell labeling. This technique, however, isnot suited for patterning the shape and size of the cells.

Accordingly, there is a need to pattern cells in a facile manner whilesubjecting the cells to relatively mild conditions.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a method for patterningcells. The method involves shielding a first portion of a surface of anarticle with a masking system. The masking system comprises a cohesivemask in conformal contact with a surface of the article. The method alsoinvolves applying an agent to a channel within the masking system to asecond portion of the surface of the article while preventingapplication of the agent to the first portion of the surface of thearticle. The method also involves applying cells onto the agent.

Another aspect of the invention provides a method for patterning cellscomprising shielding a first portion of a surface of an article with amasking system. The masking system comprises a cohesive mask inconformal contact with the surface of the article. The method furtherinvolves applying a cell-adhesion inhibitor through a channel within themasking system to a second portion of the surface of the article whilepreventing application of the cell-adhesion inhibitor to the firstportion of the surface of the article.

Another aspect of the present invention provides a method for patterningcells comprising shielding a first portion of a surface of an articlewith a masking system. The masking system comprises a cohesive mask inconformal contact with the surface of the article. The method furtherinvolves applying a cell-adhesion promoter through a channel within themasking system to a second portion of the surface of the article whilepreventing application of the cell-adhesion promoter to the firstportion of the surface of the article.

Another aspect of the present invention provides a method for patterningcells comprising providing an article having a first pattern of cells ofa first type. The method also involves applying an agent to a portion ofa surface of the article, the portion being contiguous with the firstpattern.

Another aspect of the present invention provides an article comprising afirst pattern of cells of a first type contiguous with a second patternof cells of a second type.

Another aspect of the present invention provides a method comprisingshielding a first portion of a surface of an article with a maskingsystem. The method involves allowing a cell-adhesion promoter to beapplied to a second, unshielded portion of the surface of the articlewhile preventing application of the cell-adhesion promoter to the firstportion of the surface of the article with the masking system. Themethod further involves applying a cell to the second portion of thesurface.

Another aspect of the present invention provides a method for patterningcells comprising shielding a first portion of a surface of an articlewith a polymeric masking system. The method involves applying an agentto a channel within the masking system to a second portion of thesurface of the article while preventing application of the agent to afirst portion of the surface of the article. The method further involvesapplying cells onto the agent.

Other advantages, novel features, and objects of the invention willbecome apparent from the following detailed description of the inventionwhen considered in conjunction with the accompanying drawings, which areschematic and which are not intended to be drawn to scale. In thefigures, each identical or nearly identical component that isillustrated in various figures is represented by a single numeral. Forpurposes of clarity, not every component is labeled in every figure, noris every component of each embodiment of the invention shown whereillustration is not necessary to allow those of ordinary skill in theart to understand the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of lift-off membrane (masking system)patterning to pattern cells onto a surface of an article according tothe invention;

FIG. 2 shows a schematic diagram for lift-off membrane patterninginvolving a pre-coated masking system according to the invention;

FIG. 3 shows a schematic diagram for the fabrication of a masking systemfor use in the invention;

FIG. 4 shows a photocopy of a scanning electron micrograph of a maskingsystem for use in the invention having channels shaped as holes having adiameter of about 100 μm;

FIG. 5A shows a photocopy of a fluorescence micrograph displayingcomparative results of completely coating a substrate with acell-adhesion protein followed by the addition of cells over the entireassembly;

FIG. 5B shows a photocopy of a fluorescence micrograph of the cellsadhered selectively to the surface of the substrate;

FIG. 6A shows a photocopy of a fluorescence micrograph displaying apattern of fibronectin after peeling the masking system in a process ofthe invention;

FIG. 6B shows a photocopy of a fluorescence micrograph displaying apattern of cells adhered to circular islands of fibronectin of FIG. 6A.

FIG. 7A shows a photocopy of an optical micrograph of cells patterned oncircular islands having a diameter of about 100 μm according to theinvention;

FIG. 7B shows a photocopy of an optical micrograph of cells patterned onsquare islands having a sides of a length of about 100 μm according tothe invention;

FIG. 8A shows a photocopy of a phase-contrast micrograph and afluorescence micrograph of cells patterned with a BSA pre-coatedmembrane for features having a diameter of 250 μm in a process of theinvention;

FIG. 8B shows a photocopy of a phase-contrast micrograph and afluorescence micrograph of cells patterned without a BSA pre-coatedmembrane for features having a diameter of 250 μm;

FIG. 8C shows a phase-contrast micrograph and a fluorescence micrographof cells patterned with a BSA pre-coated membrane for features having adiameter of 100 μm;

FIG. 8D shows a phase-contrast micrograph and a fluorescence micrographof cells patterned without a BSA pre-coated membrane for features havinga diameter of 100 μm;

FIG. 8E shows a phase-contrast micrograph of a surface of the membraneremoved from the process of FIG. 8B, showing attached cells; and

FIGS. 9A-D show photocopies of scanning electron micrographs displayingthe results of cell spreading after (a) 7 h, (b) 8.2 h, (c) 9.5 h, and(d) 11 h.

DETAILED DESCRIPTION

The present invention provides methods for patterning cells involving amasking system, and surfaces modified optionally using the system. Themethods are particularly advantageous in that various cell patterns canbe provided without the aid of photolithographic steps and thus patternscan be achieved in a relatively simple and inexpensive manner. Thepresent invention is applicable for patterning cells on a broad range ofsubstrates, which include most materials routinely used in cell culture.The masking system has flexibility for patterning on substrates ofessentially any shape, and has rigidity to be reused a number of times.

A resulting pattern of cells can be used for a variety of applicationsincluding observing cell growth and spreading, chemotaxis, haptotaxis,morphogenesis, and the patterning of multiple cell types. In addition,cell patterning can have long range applications in the study ofregeneration, partial regeneration or healing of human organs andwounds, i.e. tissue engineering. Other applications involve biosensors.

One aspect of the present invention provides a method for patterningcells. One method involves shielding a first portion of a surface of anarticle with a masking system. Subsequently, a second, unshieldedportion of the surface of the article is exposed to an agent such as acell-adhesion promoter, a cell-adhesion inhibitor, or a cell, before orafter removal of the masking system. The masking system can be polymericand, in one embodiment, the masking system comprises a mask having aflexible surface which allows the mask to conform to the surface. By“conform” it is meant to define essentially continuous contact betweenthe masking system and the portions of the article to be patterned. Thisembodiment is to be distinguished from, for example, a metal screen or arigid polymer, each of which can contact a surface to be masked butwhich may not be flexible enough to conformally contact the surface. Theflexibility of the mask can be provided by the use of an elastomericmask. The mask can be made of a polymeric material such aspolydimethylsiloxane (PDMS), or the like. In one embodiment, the maskcan shield selected portions of the surface by being brought intocontact with those portions. Due to the flexibility of the mask, thesurface can be either a planar or non-planar surface.

In one embodiment, there is a channel within the masking system, andpreferably a plurality of channels within the masking system. Themasking system can comprise first and second opposing surfaces where thechannel passes through the mask, connecting the first surface with thesecond surface. The channel can function to expose certain portions (asecond portion) of the surface of the article, whereas a first portionof the article is shielded due to conformal contact of the article withthe masking system. In one embodiment, the first portion is contiguouswith the second portion. In one embodiment, a channel within the mask isa hole through the mask, and by placing one surface of the mask onto asubstrate, wells are formed as defined by the walls of the channel andthe substrate surface (second portion of the surface). The mask cancontain a variety of liquid or solid agents within these wells.

In one embodiment, the mask is a polymer. A preferred polymer is apolymeric elastomer that can form a seal against the surface of thearticle. “Seal” in this context means that when the mask is sealinglyengaged with a surface and a fluid is applied to the masked surface, thefluid is allowed to contact only those portions of the masked surface inregister with channels of the mask and the fluid does not pass under themask and contact shielded portions of the article surface covered bysolid portions of the mask, so long as the fluid does not degrade themask or the surface to be patterned (in which case fluid could passunder the mask due to degradation of the mask and/or surface). Forexample, the seal can prevent a protein solution from seeping under themask. “Sealing” in this context is to be distinguished from theoperation of other rigid or flexible masks that may be brought intoconformal contact with a surface, but that can not seal against thesurface. It is a feature of the invention that masks of the inventioncan form a seal against a substrate surface in the absence of anyclamping apparatus or other apparatus used to apply a force against themask in a direction of the substrate surface. Where elastomeric surfacesare used, and the elastomeric surface and substrate surface to be maskedare clean, sealing can occur essentially instantaneously upon contactwithout application of significant pressure, and sealing can bemaintained without maintenance of any pressure. This sealing isreversible, that is, the mask can be removed from the substrate surfaceby being peeled off, and can be reused on the same or a differentsubstrate surface. Reusability of a particular mask increases with thethickness of the mask.

Exemplary techniques for fabricating a mask are described in PCTpublication WO 99/54786, entitled “ELASTOMERIC MASK AND USE INFABRICATION OF DEVICES, INCLUDING PIXELATED ELECTROLUMINESCENTDISPLAYS,” by Jackman et al., published Oct. 28, 1999, and which isincorporated herein by reference. For example, a flexible mask can becreated by a number of polymerization methods. One method, described inPCT publication WO 99/54786, involves spin-coating a pre-polymer layeronto a substrate surface having an array of cylindrical posts.

In one embodiment, the method involves applying an agent through thechannel. The method allows the agent to contact the exposed (second)portion of the surface of the article while preventing application ofthe agent to the shielded (first) portion of the article. The agent canbe applied via deposition, chemical reaction, or the like. For example,if the agent is provided as a solution, the deposition can involvespraying or dripping the solution onto the mask and through the channel,or dipping the entire substrate and masking system assembly into thesolution. In one embodiment, a vacuum may be applied to remove any airbubbles within the solution in the channel to ensure optimal surfacecoverage.

In one embodiment, the agent has physical and/or chemical propertiesthat allow its adherence to the surface of the article via adsorption.Application of agent can result in chemical reaction resulting incovalent or ionic interactions between the surface of the article andthe agent.

In one embodiment, the agent can be a cell-adhesion promoter, i.e. theagent can have physical (e.g., “sticky” materials) and/or chemicalproperties that allow cell adherence to the agent while maintaining theintegrity of the cell, and the method involves applying cells onto theagent. Cell adhesion can be achieved by specific or non-specificinteractions. Surfaces which promote non-specific interactions adheremost cells. Examples of such surfaces include ionic or charged surfaces.Hydrophilic surfaces also promote non-specific cell adhesion. An exampleof a surface involved in non-specific interactions include polymersurfaces used in biomaterials such as polylysine or plasma-treatedpolystyrene. Cell-specific interactions generally result when a cell hasa receptor which recognizes certain surfaces. For example, mammaliancells have receptors which recognize extracellular matrix proteins.Thus, cells can be patterned onto surfaces using masking systems of theinvention by first applying a cell-adhesion promoter agent to thesurface, preferably using a masking system, or applying a masking systemto a surface which already is cell-adhesion promoting. Bothcell-adhesion promoting agents and cell-adhesion promoting surfaces arewell-known in the art (some of which are described immediately above).Examples of cell-adhesion promoting agents include extracellular matrixproteins such as vitronectin, laminin, fibronectin, collagens andgelatins. Alternatively, a surface can be modified with antibodies whichrecognize certain cellular receptors. Cell-adhesion inhibiting surfacesand cell-inhibiting agents also are well-known. Examples ofcell-adhesion inhibiting agents include polyethylene glycol-basedagents. Those of ordinary skill in the art can easily screen surfacesfor their natural cell-adhesion promoting or inhibiting characteristics,or agents for cell-adhesion promotion or inhibition as follows. Variousuntreated surfaces can be studied, or various agents can be applied tosurfaces, cells can be applied to those surfaces, and the ability of thecells to adhere to the surface can be studied via morphology or othercharacteristics. This is routine for those of ordinary skill in the art.

In one embodiment, the article or surface of the article can be a metaloxide such as silica, alumina, quartz, glass, and the like orderivatives thereof, or metals such as gold, silver and copper. Thesurface can be derivatized with functional groups including amides,carboxylic acids, phosphoryl groups, hydroxyl groups, amino acid groups,amines, sulfonyl groups. Oxy compounds or plastics can also be used inaccordance with the present invention. Additional materials andfunctional groups can be found in U.S. Pat. No. 5,512,131, issued Apr.30, 1996 and incorporated herein by reference. In one embodiment, thesurface can be that of an article typically used to study cells, such asa microscope slide, petri dish, test tube or other articles. Typically,these articles are made of polystyrene, glass or polycarbonate.Functional groups discussed above, and other functionality can beprovided on the surface by coating the surface with a self-assembledmonolayer as described in U.S. Pat. No. 5,512,131. Self-assembledmonolayers are well-known and typically involve molecules each includinga group that adheres to a surface and a spacer moiety that can assemble,or pack with other spacer moieties such that when a plurality of themolecules are exposed to a surface they orient themselves in an orderedmanner with the groups that adhere to the surface against the surfaceand the spacer moieties packed relative to each other and extending fromthe surface. At the other end of each, or selected of these moleculescan be provided functional groups providing the exposed portion of theself-assembled monolayer with a desired chemical functionality.

FIG. 1 shows a schematic diagram of one example for patterning agentsassociated with cell deposition, according to the present invention.FIG. 1(a) shows an article 10 having a surface 11 with a first portion12 and a second portion 14. A masking system can comprise a mask 16.Mask 16 (shown in cross section) is brought into conformal contact withthe surface of article 10 such that the first portion 12 of surface 11is shielded. FIG. 1(b) shows the results of applying an agent 20 throughchannel 18 of masking channel 16. Because mask 16 shields second portion12, agent 20 is applied only to second portion 14 and is prevented frombeing applied to first portion 12. Agent 20 can be a cell-adhesionpromoter (e.g., fibronectin). Cells can be applied onto agent 20 onfirst portion 14 at this stage. Cells, however, will also adhere to allsurfaces coated by agent 20 (e.g., see FIG. 5A). Alternatively, mask 16can be removed prior to applying cells onto agent 20, as shown in FIG.1(c). In FIG. 1(c), substrate 10 has a pattern of agent 20 on secondportion 14, whereas first portion 12 comprises a surface free of agent20 (e.g., see FIG. 6A).

FIG. 1(d) shows the results of adding a second agent 22 to the exposedfirst portion 12. Second agent 22 can be a cell-adhesion inhibitor, suchas bovine serum albumin. Due to the inability of cells to adhere to acell-adhesion inhibitor, a cell-adhesion inhibitor functions to localizethe deposition of cells to a confined area, specifically portion 14.Generally, cells will not grow or spread onto a surface that comprises acell-adhesion inhibitor. By adding cells 24 to the surface of FIG. 1(d),a pattern of cells can be established in register with portions 14, asshown in FIG. 1(e) (e.g., see FIG. 6B).

FIG. 1 demonstrates that with techniques of the invention material canbe patterned through the holes against the substrate, and the maskremoved, leaving an array of pixels, without the requirement of stepsand apparatus involved in laser ablation, photolithography, and shadowmask procedures.

As discussed more fully below, in one embodiment cells can be depositedonto portion 14 while mask 16 is on surface 11 providing that the cellsdo not adhere to the mask or have been subjected to a pre-coatingtreatment (e.g., see FIG. 2 and discussion). The amount of cellsdeposited in each channel can depend on factors such as the diameter andheight of the channels or the density of cells in a fluid suspension.Each channel may contain one cell or several tens of cells. Anadvantageous feature of the invention is that cell(s) are confined tothe space defined by the channel until mask 16 is removed from article10. Depending on the number of cells to be deposited, the channels orholes of the mask can have a diameter of less than about 1 mm, less thanabout 500 μm, less than about 250 μm, less than about 100 μm, less thanabout 50 μm, less than about 25 μm, less than about 10 μm, less thanabout 5 μm, down to less than about 1.5 micron.

Any pattern of channels 18 in the mask, for example a pattern defined bya single channel or many channels that can be circular, oval, square,rectangular, and the like, and arranged in a grid-like array (asillustrated) or a non-array (for example random pattern) can be used.

The mask and channels can be of a variety of dimensions. In oneembodiment, the mask has a thickness of no more than about 1 mm,preferably no more than about 500 μm, more preferably no more than about200 μm, preferably no more than about 100 μm, more preferably still nomore than about 25 μm. In one embodiment, channel 18 has a preferredcross-sectional dimension 19 that corresponds to a thickness 17 of themask 20 to create a length to diameter ratio of channels of no more thanabout 5 to 1, and preferably no more than about 2 to 1. Of course, thenumber of channels and the shape of channels can be varied by any methodknown to one of ordinary skill in the art.

The conformal contact of mask 16 with article 10 should be strong enoughto prevent slippage of the mask on the article surface yet capable ofbeing removed by a peeling process. In one embodiment, the mask has athickness of at least about 50 μm. This thickness helps ensure theintegrity of the mask through several peeling processes. Preferably, thepeeling should not disturb the integrity of the pattern. It is a featureof the invention that the mask is cohesive and can be removed from asurface as a single unit and re-used, i.e., the mask facilitates a “drylift-off” procedure. The mask is cohesive in that attractive forceswithin the mask that hold the mask together are stronger than forcestypically required to remove the mask from a surface. That is, the maskcan be used to seal a surface during a deposition process, then can beremoved by lifting a portion of the mask which draws the entire maskaway from the surface, and the mask then can be reused. This is to bedistinguished from a lithographically-created mask such as a photoresistmask. Use of a cohesive mask of the invention allows formation of themask on the surface to be masked without degrading, at the surface,portions defining channels 32 (such as are degraded in creation of alithographically-created mask).

Alternatively, agent 20 is a cell-adhesion inhibitor, and upon removalof mask 16, a cell-adhesion promoter can be applied to the bare surfaceto achieve the arrangement of FIG. 1(d).

To describe FIG. 1 with a specific example, a PDMS mask (i.e., maskingsystem 16, alternatively referred to as a membrane) is used as a resistagainst the adsorption of the cell-adhesion promoter fibronectin (FN, anextracellular matrix protein) to the surface of the substrate. FNadsorbs only to the surface of the substrate that is exposed by thepores of the membranes (see FIG. 6). Removal of the mask from thesurface generates a pattern of FN. The substrate is then exposed tobovine serum albumin (BSA)-containing culture media to ensure that theremainder of the surface is coated by a protein that resists cellattachment. Cells from a suspension adhered to this substrate only inthe pattern defined by the pores of the membrane (see FIG. 7). FIG. 1 isnot drawn to scale and FIG. 1 does not imply that layers of BSA and FNhave the same thickness. The mask features may be curved at the top as aresult of menisci formation during spin coating or other processes.

For certain cell types, it may be preferable to pattern the cells byconfining the cells within mask channels. The mask channels provide aphysical barrier to contain and thus maintain control of cell size andshape, or the size and shape of a layer of cells. In this embodiment,ideally, a mask is positioned on the surface to create certain wells asdefined by mask channels and the surface, and cells are deposited intothese wells. Because the mask itself may have cell-adherent properties,however, removal of the mask may result in tearing of cell walls in someinstances, particularly where cells are adhered simultaneously to themask and the substrate. Accordingly, to ensure that contacting the cellswith the mask will not damage cell walls upon mask removal, in oneembodiment, a cell pattern is provided by use of a pre-coated mask. Atleast a portion of the mask, preferably those portions that couldcontact cells in the process, can be pre-coated with an agent that is acell-adhesion inhibitor, such as bovine serum albumin. Thus, a cell doesnot have a tendency to adhere to the mask and peeling off the mask doesnot damage the cells.

FIG. 2 shows an example for patterning cells involving a pre-coatingtreatment of the masking system. FIG. 2(c) shows an article 30 having asurface 31 comprising a first portion 32 and a second portion 34. Mask36 (shown in cross section) shields first portion 32 by being inconformal contact with first portion 32 whereas channels 38 exposesecond portion 34. FIG. 2(c) also shows a coating of a first agent, acell-adhesion inhibitor agent 40 which has been applied only to exposedsurfaces of mask 36 and not on exposed surfaces of second portion 34 ofarticle 30. Addition of a second agent 42, such as a cell-adhesionpromoter, provides a coating over second portions 34 of article 30, asshown in FIG. 2(d). Preferably, agent 40 is selected to resistadsorbtion of agent 42. The addition of cells 43 results in celladhesion on agent 42 only, and cell adhesion is inhibited on surfacescovered by agent 40, as shown in FIG. 2(e) (e.g., see FIG. 5B). Thearrangement shown in FIG. 2(e) provides the advantage that upon peelingthe masking system 36 from article 30, the cell-adhesion inhibitornature of the surface of article 36 coated with agent 40 will reduce anyfriction between masking system 36 and the cells, thus promoting cellintegrity (e.g., see FIG. 8).

In this embodiment, article 36 can be pre-coated with a cell-adhesioninhibitor 42 as shown in FIGS. 2(a) and (b). In FIG. 2(a), a firstsurface 37 of mask 36 is contacted with a surface 46 of substrate 44.Preferably, first surface 37 is brought into conformal contact withsurface 46. FIG. 2(b) shows the results of coating agent 40 (acell-adhesion inhibitor) onto mask 36 and substrate 44. Surface 37 ofmask 36 is free of the agent 40. Removal of mask 36 from substrate 44followed by placement of mask 36 on surface 31 of article 30 results inunexposed surfaces (second portions 34) of article 30 free of agent 40.Subsequently, mask 36 can be used to shield portions of article 30, asshown in FIG. 2(c).

FIG. 2(f) shows the results of removing mask 36 from article 30,exposing first portions 32, followed by application of agent 50 (eithera cell-adhesion inhibitor or promoter) to first portions 32. Where agent50 is a cell-adhesion promoter, the cells applied onto agent 42 can beallowed to spread onto agent 50, the results of which are shownschematically in FIG. 2(h). Thus, the invention provides a novel mediumto study the effects of cell spreading, or other cellular phenomena,such as chemotaxis, haptotaxis or morphogenesis from a predeterminedarea (i.e., shape and size) of an individual cell to groups of cells.

It can be seen that in this aspect of the invention, a method isprovided for a simple and inexpensive method to grow attached cellswithin patterned constraints. In one embodiment, the constraints can bereleased to allow the cells to spread. Most current techniques forpatterning cells are not directly compatible with a process thatrequires the cells to be grown within patterned constraints, and thenreleasing those constraints and allowing the cells to migrate.Patterning of cells is an experimental tool that can be useful, forexample, for studying and controlling the behavior ofanchorage-dependent cells. Patterning of cells has been previouslyachieved with microcontact printing (see for example, C. S. Chen et al.Science 1997, 276, 1425-1428; R. Singhvi et al. Science 1994, 264,696-698; Prog. 1998, 14, 378-387; G. P. Lopez et al. J. Am. Chem. Soc.1993, 115, 5877-5878; A. Kumar et al. Appl. Phys. Lett. 1993, 63,2002-2004; M. Mrksich et al. Trends Biotech. 1995, 13, 228-235).Although microcontact printing is an experimentally convenient techniquethat has sufficient resolution to allow the patterning of single cells,in its simplest configuration it does not allow the cells to be“released” from the pattern; that is, once a pattern of SAMs has beenformed, fibronectin adsorbed, and cells attached, there is no practicalway of changing the pattern or allowing the cells to spread beyond theboundaries of this pattern. Other patterning methods involve morecomplex processes.

In another embodiment, agent 42 is a first cell-adhesion promoter andagent 50 is a second cell-adhesion promoter. This embodiment provides amethod for patterning multiple cell types onto a single substrate inwhich cells of a second type can be applied onto agent 50. In oneembodiment, the first cell-adhesion promoter is specific for cells 43 ofa first type and the second cell-adhesion promoter is specific for cells45 of a second type (FIG. 2(g)).

FIG. 2 can be described with reference to a specific example. The mask36 is pre-coated with agent 40, namely BSA, selectively on one of itssides and in interior channel surfaces and the pre-coated mask is usedduring the adsorption of FN to a clean substrate surface (FIG. 2 d).Cells adhere to the surface of the substrate that is coated with FNwhile being prevented from adhering to the walls of the membranechannels or the top of the mask, coated with BSA. Accordingly, uponpeeling, the mask does not damage the cells that remain attached to thesurface of the substrate in the pattern defined by the holes of themembrane (see FIG. 2F, FIG. 8). Removal of the mask exposes a pattern ofcells adjacent exposed portions of the surface. The protected areas ofthe substrate can then be modified by the adsorption of an adhesiveprotein that allows the patterned cells to spread to the exposedsurface. Alternatively, another cell type can be adhered to the surface.Upon removal of mask 36, cell integrity can be tested via a fluorescenceassay. In this assay, cells are incubated with a dye (e.g., propidiumiodide) which diffuses only into cells which have damaged membranes andwhich become more fluorescent upon complexing with DNA.

Alternately, the mask can be prepared of a material that does not adherecells. Thus, a pre-coating step is unnecessary. The material of thenon-adherent mask can depend on the cell-type.

More than two cell types can be patterned on a single surface, bycontrolled shielding of various portions of the article. As described inPCT publication WO 99/54786, masking systems involving multiple maskscan be used in differing overlaying arrangements to control theapplication of particular agents or cell types into desired portions ofthe surface.

It is a feature of the invention that mask/surface systems providemethods for observing cell growth when cells are initially depositedwithin a physically-constrained barrier. Cells can be grown within thewells as defined by mask channels and the substrate surface. Surfacechemistry of the mask walls and substrate surface can be controlled in away to cause cells to attach and spread on the substrate but not attachto the mask. The mask can then be removed to allow cells to spread ontothe rest of the surface.

Another advantageous feature of the invention is the provision ofchannels that are of sufficiently small size to control the size andshape of a single cell. The physical constraints can be used to inhibitcell growth while the mask is conformed to the substrate andsubsequently promote cell growth upon removal of the mask from thesubstrate. Known techniques for studying cell spreading and migrationtypically have not involved controlling the shape and size of cellsbefore allowing them to spread and migrate. The shape and size of cellsdetermines their passage through the cell cycle. It is known that a cellgrowth involves a cycle of stages. A cell may not attain the next stageuntil it has reached a certain size. The size and shape of cells may notonly affect their ability to spread, but ultimately the ability tomigrate about a surface. The ability to control cell growth andmigration has many applications in the control of wound healing, celldeath (apoptosis) and differentiation. Angiogenesis (capillary growth)is one example of the differentiation of bovine capillary endothelialcells. A channel can be used to constrict the cell to a specific size,whereupon removal of the channel results in cell growth and thus thecontrol of cell migration about the substrate surface or a portion ofthe substrate surface.

It is to be understood that the order of steps for shielding via themasking system, application of agents and application of cells can bevaried to obtain a desired result. For example, another aspect of theinvention provides a method for patterning cells, comprising shielding afirst portion of a surface of an article with a masking systemcomprising a cohesive mask in conformal contact with the surface of thearticle. The method involves applying a cell-adhesion inhibitor througha channel within the masking system to a second portion of the surfaceof the article while preventing application of the cell-adhesioninhibitor to the first portion of the surface of the article. Referringback to FIG. 1, this aspect presents a different result from thatdescribed previously for FIG. 1(c), namely second portions 14 having acell-adhesion inhibitor applied thereon, and exposed first portions 12.This aspect describes a different method for obtaining the result shownin FIG. 1(d).

Another aspect of the invention provides a method for patterning cells,comprising providing an article having a first pattern of cells of afirst type and applying an agent to a portion of the surface of thearticle. This portion can be contiguous with the first pattern. In oneembodiment, the agent can be a cell-adhesion inhibitor for cells of thefirst type. In another embodiment, the agent is a cell-adhesion promoterfor cells of a second type. This method is advantageous in thepatterning of multiple cells or for methods allowing cell spreading,where the affinity of the different cell-adhesion promoters is notstrong enough to differentiate between cell types to a desired extent.Thus, by adhering cells of a first type to the first cell-adhesionpromoter prior to applying the second-adhesion promoter onto thesurface, greatly differing affinities of different cell-adhesionpromoters is not as critical a requirement to provide discrete patternsof multiple cell types.

The ability to pattern multiple cell types has applications in organregeneration. For example, it is known that certain organ types havestriated patterns of different cell types. For example, a surface mayhave one continuous portion of cells of a first type adjacent acontinuous portion of cells of a second type which in turn is anadjacent another portion of cells of a first type or even a third type.The continuous portion may resemble a layer of any shape runningparallel to the substrate surface. Thus, there is a need to control thepositioning of cells of a first type with respect to cells of a secondtype. For some cases, the cells of the first type do not have greatlydiffering affinities for surface adherence than the cells of the secondtype. Such close affinities may present strategic difficulties in thatone surface (or agent on the surface) can adsorb significant quantitiesof cells of either type. The flexible mask of the present inventionshields certain portions of the substrate surface indiscriminate of cellaffinities, and thus, the need to fine-tune cell-substrate affinity iscircumvented. For the above example, the striated layers of cells can beprovided by a mask having channels shaped to have an extremely longlength but short widths. Of course, the shape of the channels do nothave to resemble a regular geometrical shape, and can be of any shapefeasible that can withstand the coating, depositing and peelingprocesses.

Another aspect of the present invention provides an article comprising afirst pattern of cells of a first type contiguous with a second patternof cells of a second type. This aspect is to be distinguished from arandom array of cells of multiple types. The article can have more thantwo patterns of different cell types by using the methods describedherein.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples below. Thefollowing examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.

GENERAL CONDITIONS

Materials. SU-8 50 photoresist was supplied by Microlithography ChemicalCorp. (Newton, Mass.). We used rigid chrome masks (AdvancedReproductions, North Andover, Mass.) or transparencies as the photomasksin the photolithographic step. Poly(dimethylsiloxane) (PDMS); Sylgard184) was obtained from Dow Corning (Midland, Mich.). Bacteriological andtissue culture grade Petri dishes were purchased from Falcon. No. 2glass slides from Corning Inc. (Corning, N.Y.) were used as received.Silicon wafers <110> were obtained from Silicon Sense Inc. (Nashua,N.H.), and were also used as received. Phosphate buffered saline packetswere purchased from Sigma and diluted to the desired concentration (150mM, pH=7.4) with distilled water. Dulbecco's modified eagle medium(DMEM), BSA (fraction V), and fibronectin were purchased from Gibco(Life Technologies, Rockville, Md.); we added 5 μM HEPES (JRHBiosciences, Lenexa, Kans.) to the medium. Sodium dodecyl sulfate (SDS)was purchased from Bio Rad (Hercules, Calif.). Gelatin was purchasedfrom DIFCO Laboratories (Detroit, Mich.). Para-formaldehyde waspurchased from Electron Microscopy Sciences (Ft. Washington, Pa.).

Substrates. We patterned cells on the surfaces of Petri dishes, PDMS,glass slides, silicon (<110>, native oxide). Unless specified otherwise,we always use Petri dishes as the substrates.

EXAMPLE 1 Fabrication of Masking System

Fabrication of Patterned Photoresist Structures and Membranes. Withreference to FIG. 3, arrays of cylindrical posts of photoresist 60 werefabricated on silicon wafers 62 using standard photolithographictechniques and rigid chrome masks. Arrays of square features werefabricated using transparencies as photomasks. We used procedureswell-known in the art to fabricate features there were 50 μm high.

Fabrication of Masking Systems. Elastomeric polymer membranes werefabricated using the procedure described by Jackman et al. (Jackman etal., Langmuir, vol. 15, pp. 2973-2984, (1999)). The PDMS prepolymer 64(mixed in a 10:1 ratio with a crosslinking catalyst) was spin-coated onthe bas-relief of patterned photoresist using parameters known toproduce a film that was thinner than the height of the features ofphotoresist. For features that were 50 μm tall, we spin-coated PDMSprepolymer at 3000 rpm for 60 sec to generate a film that wasapproximately 45 μm thick. The PDMS films were cured for 2 h at 60° C. Athick layer of PDMS prepolymer was added to the edges of the membranesin dropwise fashion; after curing, this layer of PDMS provided a framethat would support the substrates; we typically used pieces that were2×2 cm. The films were kept at 60° C. overnight. Prior to use in cellculture, we removed low molecular weight polymer from the membranes bysoaking them in dichloromethane for 12 h. The membranes were then soakedin ethanol for 1 hour and dried in an oven at 60° C. for 12 hours. Themembranes 66 were removed from their supports (photoresist posts,cylindrical or square, on silicon) using tweezers and they were then cutto the desired sizes along the edges of the support. FIG. 4 shows aphotocopy of a scanning electron micrograph of a membrane with 100 μmcircular holes. The membrane is approximately 50 μm thick. The membraneis curved upwards while being removed from the surface as in the peelingstep in FIGS. 1 and 2. This image illustrates the elastomeric propertiesof the membrane. The membranes generally come into conformal contactwith the substrates 68. In cases when the membranes were not flat on thesurface and adhered to themselves, we placed a drop of ethanol on themto facilitate the formation of a flat seal. The ethanol wets the surfaceof the membrane preferentially and it allows it to become flat;evaporation of the ethanol leaves the membranes flat on the substrates.The membranes were ready for use after evaporation of the ethanol.

For biological experiments, low molecular weight organic substances canbe extracted from the membranes by soaking the membranes indichloromethane for several hours (overnight) followed by drying at 60°C. for several hours (overnight). The membranes are then placed ontoPetri dishes and covered with a few drops of absolute ethanol. Ethanolcan help decrease the tendency of the membranes to adhere to themselvesand facilitate a formation of conformal contact between the membrane andthe surface of the substrate. Ethanol also sterilizes the membranes andthe surfaces of the substrates. Alternatively, a conformal seal with asubstrate can be achieved when the membrane is positioned on thesubstrate in the presence of PBS buffer. This buffer also affordsmaintaining the hydration of the layer of BSA. A layer of BSA that hasbeen allowed to dry does not resist the attachment of cells as well asone that has been kept hydrated. In the Examples, substrates are exposedto suspensions of bovine capillary endothelial (BCE) cells. Typically a2 mL suspension of 25,000 cells/mL in a dish having an area of 962 mm².

Procedure used to wash the membrane after use in cell culture. Themembranes were kept in buffered SDS (10 mg/mL, PBS at pH −7.4) for 30min at room temperature and 30 min at 90° C., followed by extensiverinsing with deionized water and ethanol. The membranes were thenextracted with dichloromethane for 12 hours and dried at 60° C. for 12hours. These membranes (like other microscopic structures made of PDMS)can also be autoclaved for 20 min (121° C., 115 kPa).

EXAMPLE 2 Surface Modification

a) Pre-coating the membrane with a cell adhesion inhibitor. In a laminarflow hood, the membranes were placed on the surface of a sterile Petridish with a few drops of ethanol. The liquid sterilized the membranes bykilling bacteria. Drops of a buffered solution of BSA (1% w/v, in PBS orDMEM at pH=7.4) were placed on the membrane to cover the holes, in amanner schematically described in FIG. 2. Since the liquid did not fillthe hydrophobic pores, vacuum was applied (˜30 sec) and released (˜500mTorr) twice to extract the air trapped in the pores. BSA was allowed toadsorb to the surfaces for 15 min. The substrates were then rinsed threetimes with PBS; the membranes were peeled from the support in thepresence of PBS, and transferred to a clean Petri dish covered with PBSto help seal the membrane onto the dish.

b) Patterning Proteins on Substrates. Drops of a cell-adhesion promoter,buffered fibronectin (50 μg/mL, PBS with pH=7.4) or gelatin (1.5% w/v,PBS with pH=7.4) solutions were placed on a membrane adhered to asubstrate. Vacuum was applied (ca. 30 sec) and released twice to extractthe air trapped in the pores. The protein was allowed to adsorb to thesurfaces for 1 hr (in the case of fibronectin) or for 15 min (in thecase of gelatin). The assembly of the membrane and substrate was thenrinsed with buffer 3 times. The membrane was removed from the surfacewith a pair of tweezers, in the presence of culture media that contained1% (w/v) BSA. After 15 min, fresh media was introduced into the dish,followed by a suspension of cells.

Immunofluorescent staining of adsorbed FN. The substrates coated with FNwere exposed to 4% (v/v) PFA in PBS buffer (pH=7.4) for 20 min, and thenimmersed in a solution of rabbit anti-human fibronectin IgG (Sigma, 5μg/mL) for 1 hour. The substrates were rinsed twice with PBS containing0.1% (w/v) BSA and 0.1% (v/v) Triton X-100, and placed in contact with100 μL of Texas Red®-labeled goat anti-rabbit IgG (Amersham LifeSciences, 50 μg/mL) for 1 hour; the samples were then rinsed, and sealedonto microscope slides with Fluoromount-G (Southern Biotechnology,Inc.).

EXAMPLE 3 Cell culture

a) Growth and attachment. Bovine adrenal capillary endothelial (BCE)cells were cultured under 10% CO₂ on cell culture Petri dishes (Falcon)coated with gelatin in DMEM containing 10% calf serum, 2 mM glutamine,100 μg/mL streptomycin, 100 μg/mL penicillin, and 1 ng/mL basicfibroblast growth factor (bFGF).² Prior to incubation with the patternedsubstrates prepared using MEMPAT, cells were dissociated from cultureplates with trypsin-EDTA and washed in DMEM containing 1% BSA(BSA/DMEM). The suspension of cells (typically 25,000 cells/mL, 2 mLtotal volume) was placed on the substrates in chemically defined medium(10 μg/mL high density lipoprotein, 5 μg/mL transferrin, 5 ng/mL basicfibroblast growth factor in 1% BSA/DMEM) and incubated in 10% CO₂ at 37°C.

b) Fixing and Staining Cells. Substrates that contained cells were fixedwith PFA for 20 min and washed with PBS. The substrates were then washedwith methanol for 1 min, and stained with Coomassie Blue (5 mg/mL in 40%v/v methanol, 10% v/v acetic acid, and 50% v/v water) for 30 sec; theywere then rinsed with distilled water and dried in air.

Procedures Used to Study Cell Spreading. Cells were allowed to attach topatterns of gelatin or FN defined by the holes of the BSA-coatedmembranes. After 7-24 hours, the assembly defined by the membrane, thesubstrate, and the attached cells was rinsed with PBS buffer three timesto remove BSA from the solution and it was then immersed in a PBSsolution of gelatin (1.5% w/v). The membrane was peeled gently from thesurface with a pair of tweezers and the substrates were incubated for 15min to adsorb gelatin on the areas of the surface that were protected bythe membrane. The substrates were then rinsed once with culture medium(DMEM) before being placed in the incubator for ca. 4 hours, to allowthe cells to spread onto the previously protected areas of thesubstrates.

Characterization of Damage to Cells. Membranes were gently removed fromsubstrates that presented attached cells. The attached cells wereincubated with a solution of propidium iodide in culture medium (10μg/mL) for 15 minutes. The cells were imaged with a fluorescencemicroscope immediately after rinsing the samples twice with culturemedium at 37° C. The intensity of the fluorescence of propidium iodidedecreased as the dye diffused out of the cells over the course of twohours; this diffusion into the medium also decreased the contrastobtained in the micrographs.

Microscopy. a) Phase contrast and fluorescence microscopy were performedwith a Nikon Axiophot equipped with a 35 mm camera. The developednegatives or slides were scanned into a digital format with a NikonLS-400 slide scanner. Images were processed only by performingoperations uniformly on the entire image; we typically converted thecolor images to black and white and enhanced the contrast to ensure thatthe fine features of the cell structure would appear in the version ofthe figure printed in the journal.

b) SEM micrographs were obtained on a JEOL JSM-6400 scanning electronmicroscope operating at 15 keV.

EXAMPLE 4 Comparison of Pre-Coating Masking System With Cell-AdhesionInhibitor Verses No Pre-Coating

FIGS. 5A and 5D show photocopies of optical micrographs displaying theresult of coating the membranes with different proteins. This processhelps to determine whether cells attach to the substrate and themembranes or only to the substrate. FIG. 5A shows cells that are adheredover the entire assembly of membrane and substrate that was coated withFN as described above without use of a cell-adhesion inhibitor coatingof the masking system. FIG. 5B shows that cells adhere selectively tothe surface of the substrate that was coated with FN using a membranethat is pre-coated with BSA (see FIG. 2). The cells do not attach to themembrane.

EXAMPLE 5

FIG. 6A shows a photocopy of a fluorescence image displaying a patternof FN generated on a bacteriological petri dish using the masking systemtechnique described above, following the application and release ofvacuum. After an incubation of 1 hour followed by three rinsing steps,the membrane is removed from the substrate in the presence of culturemedium that contained BSA or any other BSA-containing solution (seeExample 1). Thus, the membrane is removed after the adsorption ofprotein and before attachment of cells. This method provides a patternof cells while exposing the rest of the surface for the adsorption ofanother agent, e.g., a protein (see FIG. 7). The FN pattern on thesurface is incubated with fluorescently labeled antibodies that make theFN appear light gray in fluorescence microscopy. FIG. 6B shows a patternof cells adhered to circular islands of FN with 50 μm in diameter,prepared with the same method as for FIG. 6A.

EXAMPLE 6

FIGS. 7A and 7B show photocopies of optical micrographs of cellspatterned on a bacteriological petri dish that presented islands of FNthat were generated using the technique of Example 2. The membrane iscoated with BSA and then placed on a clean petri dish and exposed to asolution of FN (50 mg/mL in PBS) as described in Example 2. The membraneand the substrate are covered with a suspension of cells for 24 hours.The membrane is removed and the cells are fixed and stained to show thenuclei and parts of the cytoskeleton. The efficiency of patterning iscomparable to that achieved with MEMPAT (see FIG. 6). FIG. 7A showscells patterned on circular islands 100 μm in diameter. FIG. 7B showscells patterned on square islands with 100 μm sides.

EXAMPLE 7

FIG. 8 shows the results of pre-coating membranes with BSA to avoiddamage to the membranes of cells during membrane removal, versus nopre-coating. A membrane is placed in conformal contact with a substratesurface and the membrane and the surface of the substrate are coatedwith fibronectin (FN). A suspension of BCE cells is then placed incontact with such a surface and the membrane is removed. Cells adhere tothe substrate specifically or to the substrate and the membrane. After24 hours in culture, the membranes are removed from both types ofsamples and the cells that remain attached to the surface of thesubstrate are incubated with propidium iodide dissolved in culturemedium (10 μg/mL), for 15 minutes; this fluorescent agent onlypenetrates the membranes of damaged cells. The samples are imaged afterrinsing with culture medium.

Micrographs on the left-hand side of FIG. 8 are obtained byphase-contrast microscopy. Micrographs on the right-hand side of FIG. 8are obtained by fluorescence microscopy. For the fluorescencemicrographs, cells are incubated with propidium iodide after membraneremoval.

FIGS. 8A and 8C show BCE cells patterned on a substrate through amembrane which was pre-coated with BSA using the procedure of Example 2.After removal of the membrane, the cells were incubated with propidiumiodide. FIG. 8A shows a pattern of features having a diameter of 250 μmwhereas FIG. 8C shows a pattern having features of a diameter of 100 μm.The corresponding fluorescence micrograph show that no cells internalizethe fluorescent dye indicating that the cell membranes were not damaged.

FIGS. 8B and 8D show the results of a membrane and substrate coated withfibronectin where the membrane was not pre-coated with BSA, as describedin Example 1. FIG. 8B shows a pattern having features of a diameter 250μm whereas FIG. 8D shows a pattern having features of a diameter 100 μm.Removal of the membrane in FIGS. 8B and 8D show a poorly defined patternof cells. Many of the adhered cells appear to be damaged in thecorresponding fluorescence micrographs.

FIG. 8E shows a surface of a membrane that was used in FIG. 8B afterremoval of the membrane from the surface, where the surface of themembrane is covered by attached cells. Many cells also adhere to thewalls of the holes. A fluorescence micrograph of the membrane revealedthat many of the cells attached in the holes presented damagedmembranes.

EXAMPLE 8

This example provides a demonstration that the techniques of theinvention allow the study of cell spreading. Cells are patterned onpetri dishes using BSA-coated membranes as described in Example 2. Thecells were allowed to spread on the substrate in conformal contact witha membrane for 6-20 hours until the cells covered the entire areaexposed by a channel of the membrane. The membrane was removed from thesubstrate in the presence of a solution of gelatin (free of all BSA) andincubated for 20 minutes. This procedure coats the areas of thesubstrate that had previously been covered by the membrane with a layerof adhesive protein. After replacing the solution of gelatin withculture medium, the cells were incubated. For varying intervals over aperiod of 7 hours to 11 hours and at each indicated time from thebeginning of the experiment, one sample is fixed and stained. FIGS. 9A-Dshow photocopies of scanning electron micrograph images displaying anarea that is representative of the entire sample. FIG. 9A shows adiscrete pattern of cells. As the cells are allowed to spread as shownin FIGS. 9B and 9C, eventually the cells spread over the entire portionof the substrate as shown in FIG. 9D.

Those skilled in the art would readily appreciate that all parameterslisted herein are meant to be examples and that actual parameters willdepend upon the specific application for which the methods and apparatusof the present invention are used. It is, therefore, to be understoodthat the foregoing embodiments are presented by way of example only andthat, within the scope of the appended claims and equivalents thereto,the invention may be practiced otherwise than as specifically described.

1. A method for patterning cells, comprising: shielding a first portionof a surface of an article with a masking system comprising a cohesivemask in conformal contact with the surface of the article; applying anagent through a channel within the masking system to a second portion ofthe surface of the article while preventing application of the agent tothe first portion of the surface of the article; and applying cells ontothe agent.
 2. The method of claim 1, wherein the masking systemcomprises a flexible mask including a first surface and an opposingsecond surface, and the channel is one of a plurality of channelspassing through the mask and connecting the first surface with thesecond surface.
 3. The method of claim 2, further comprising removingthe masking system from the surface of the article.
 4. The method ofclaim 1, wherein the agent is a cell-adhesion promoter.
 5. The method ofclaim 2, further comprising removing the masking system from the surfaceof the article prior to the step of applying cells onto the agent. 6.The method of claim 5, wherein the agent is a first agent and the methodfurther comprises adding a second agent to the first portion of thesurface.
 7. The method of claim 6, wherein the first agent is acell-adhesion promoter.
 8. The method of claim 7, wherein the firstagent is a protein.
 9. The method of claim 8, wherein the protein isfibronectin.
 10. The method of claim 6, wherein the second agent is acell-adhesion inhibitor.
 11. The method of claim 1, wherein the firstportion of the surface of the article is contiguous with the secondportion.
 12. The method of claim 2, wherein the agent is a first agentand the method comprises pre-coating at least a portion of the maskingsystem with a second agent prior to the shielding step.
 13. The methodof claim 12, wherein the pre-coating step comprises: contacting thefirst surface of the masking system with a substrate; and coating thesecond agent onto the second surface and the plurality of channels ofthe masking system, wherein the first surface of the masking system isfree of the second agent.
 14. The method of claim 13, wherein theshielding step comprises: removing the masking system from thesubstrate; and bringing the first surface of the masking system intoconformal contact with the first portion of the surface of the article.15. The method of claim 14, wherein the first agent is a cell-adhesionpromoter.
 16. The method of claim 15, wherein the second agent is acell-adhesion inhibitor.
 17. The method of claim 16, further comprisingremoving the masking system from the surface of the article prior toapplying the cells onto the first agent.
 18. The method of claim 16,further comprising removing the masking system from the surface of thearticle.
 19. The method of claim 18, further comprising adding a thirdagent to the first portion of the surface of the article.
 20. The methodof claim 19, further comprising allowing the cells applied to the firstagent to spread onto the third agent.
 21. The method of claim 19,wherein the first agent is a first cell-adhesion promoter and the thirdagent is a second cell-adhesion promoter.
 22. The method of claim 21,further comprising adding cells of a second type to the third agent. 23.The method of claim 1, wherein the channel has a dimension forcontrolling the growth of a single cell. 24-34. (canceled)
 35. A methodfor patterning cells, comprising: providing an article having a firstpattern of cells of a first type; and applying an agent to a portion ofthe surface of the article, the portion being contiguous with the firstpattern.
 36. The method of claim 35, wherein the agent is acell-adhesion inhibitor for cells of the first type
 37. The method ofclaim 35, wherein the agent is a cell-adhesion promoter for cells of asecond type.
 38. The method of claim 37, further comprising applying thecells of the second type onto the agent.
 39. The method of claim 35,wherein the providing step further comprises: bringing a masking systemin conformal contact with the portion of the surface of the article; andapplying a cell-adhesion promoter through a channel within the maskingsystem.
 40. The method of claim 39, wherein the providing step furthercomprises pre-coating the masking system with a cell-adhesion inhibitorprior to the step of bringing the masking system in conformal contactwith the surface of the article.
 41. An article comprising: a firstpattern of cells of a first type contiguous with a second pattern ofcells of a second type. 42-47. (canceled)